Improved Medium for Enumeration of Clostridium perfringens

An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.     

Sporulation of Clostridium perfringens in a Modified Medium and Selected Foods

A modified sporulation medium for Clostridium perfringens was formulated in which a larger number of spores were produced than in SEC broth and in which spores of greater heat resistance were produced than in Ellner's medium when it was also used as the suspending medium. This modified medium consisted of 1.5% peptone; 3.0% Trypticase; 0.4% starch; 0.5% NaCl; and 0.02% MgSO(4). The addition of 0.1% sodium thioglycolate and 0.0001% thiamine hydrochloride was optional. The optimal temperature for sporulation of five strains was 37 C in comparison with 5, 22, and 46 C. Sporulation had occurred by 6 hr and was essentially complete after 20 hr at 37 C. Noyes veal broth without glucose also supported the formation of heat-resistant spores but in smaller numbers than did the modified medium. Very low numbers of spores, or none, were produced under the same conditions in pea or tuna slurries.     

Rapid Technique for the Enumeration of Clostridium perfringens

A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar.     

Comparison of Media for the Enumeration of Clostridium perfringens

For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 10(4) organisms per g, but with 10(6) organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested.     

Quantitation of Clostridium perfringens in Foods

A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks.     

Evaluation and Modifications of Media for Enumeration of Clostridium perfringens

The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose.     

Incidence of Clostridium perfringens in American Foods

Food samples were examined for the presence of Clostridium perfringens. A medium described by Mossel and later modified by Angelotti et al. was used for the detection and enumeration of C. perfringens. The incidence of C. perfringens observed in the foods examined was 6.1%. C. perfringens was recovered from 2.7% of the commercially prepared frozen foods, 3.8% of fruits and vegetables, 5.0% of spices, 1.8% of home-prepared foods, and 16.4% of raw meat, poultry, and fish.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Chemically Defined Medium for the Growth of Clostridium perfringens

A defined medium which supports growth of Clostridium perfringens at low inoculum levels was developed. Generation time for strain 8797 was 1.5 times greater than previously reported for growth in purged fluid thioglycolate medium.     

Enumeration of Food-Borne Clostridium perfringens in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes.     

Medium for the Selective Enumeration of Lactic Acid Bacteria from Foods

A nitrite actidione polymyxin agar was developed for the enumeration of lactic acid bacteria. It was effective in recovering organisms from pure cultures and from foods.     

Chemically Defined Medium for Growth and Sporulation of Clostridium perfringens

A chemically defined medium was developed that could support sporulation and growth of Clostridium perfringens strains ATCC 12916 and H9. This medium consisted of a modification of the basal medium of Boyd et al. plus 0.1% sodium thioglycolate and 0.5% monosodium glutamate. Five other strains grew, but did not sporulate, in this medium. With the addition of more vatamins into the medium, two more strains grew but did not sporulate. The effects of glucose, monosodium glutamate, ammonium glutamate, and sodium thioglycolate on growth and sporulation of C. perfringens ATCC 12916 in the defined medium was investigated.     

Enumeration of Fecal Clostridium perfringens Spores in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin.     

Medium for Toxin Production by Clostridium perfringens in Continuous Culture

A tryptone-salts medium for continuous growth and alpha toxin production of Clostridium perfringens type A in an adapted chemostat is described. In such steady-state cultures, fermentative and biochemical activity of C. perfringens remained unchanged. Toxigenic ability to produce alpha, theta, and nu toxins was preserved.     

New Medium for the Isolation and Enumeration of Pseudomonads

A new medium containing 200 ppb of 2-hydroxy-2',4,4'-trichlorodiphenyloxide (CH3565) and 10 ppm of cetyl-trimethyl-ammonium bromide (Cetrimide) in tryptic soy agar was developed and tested with 19 pure cultures of Pseudomonas, 20 microorganisms of other genera, commercially prepared ground beef, and laboratory-prepared inoculated ground beef. The new medium, CETCH agar, was compared with an antibiotic-containing medium. CETCH agar provided greater pseudomonad recoveries, a shorter incubation period prior to plate counting, and greater ease of preparation.     

Comparison of Sulfite-Polymyxin-Sulfadiazine Medium and Tryptose-Sulfite-Cycloserine Medium Without Egg Yolk for Recovering Clostridium perfringens

The overall recoveries of spores and of actively growing, heat-stressed, coldshocked, and frozen cells of five strains of Clostridium perfringens were significantly greater (95% confidence limits) on tryptose-sulfite-cycloserine medium without egg yolk than on sulfite-polymyxin-sulfadiazine medium.     

Spoilage Bacteria in Canned Foods: II. Sulfide Spoilage Bacteria in Canned Mushrooms and a Versatile Medium for the Enumeration of Clostridium nigrificans

Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established.     

Improved Solid Medium for the Detection and Enumeration of Pectolytic Bacteria

An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.